Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to produce plants which have unique characteristics of agronomic importance. Certainly, one such advantageous trait is more cost effective, environmentally compatible weed control via herbicide tolerance. Herbicide-tolerant plants may reduce the need for tillage to control weeds thereby effectively reducing soil erosion.
One herbicide which is the subject of much investigation in this regard is N-phosphonomethyl-glycine commonly referred to as glyphosate. Glyphosate inhibits the shikimic acid pathway which leads to the biosynthesis of aromatic compounds including amino acids and vitamins. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).
It has been shown that glyphosate tolerant plants can be produced by inserting into the genome of the plant the capacity to produce a higher level of EPSP synthase which enzyme is preferably glyphosate tolerant (Shah et al., 1986). The introduction into plants of glyphosate degradation gene (s) could provide a means of conferring glyphosate tolerance to plants and/or to augment the tolerance of transgenic plants already expressing a glyphosate tolerant EPSP synthase depending upon the physiological effects of the degradation products.
Glyphosate metabolism (degradation) has been examined in a wide variety of plants and little degradation has been reported in most of those studies. In those instances where degradation has been reported, the initial breakdown product is usually aminomethylphosphonate (AMPA) (Coupland, 1985; Marshall et al., 1987). In these instances, it is not clear if glyphosate is metabolized by the plant or the contaminating microbes on the leaf surface to which glyphosate was applied. AMPA has been reported to be much less phytotoxic than glyphosate for most plant species (Franz, 1985) but not for all plant species (Maier, 1983; Tanaka et al., 1988). Glyphosate degradation in soils is much more extensive and rapid (Torstensson, 1985). The principal breakdown product identified is AMPA (Rueppel et al., 1977; Nomura and Hilton, 1977); a phosphonate that can be metabolized by a wide variety of microorganisms (Zeleznick et al., 1963; Mastalerz et al., 1965; Cook et al., 1978; Daughton et al., 1979a; 1979b; 1979c; Wackett et al., 1987a). A number of pure cultures of bacteria have been identified that degrade glyphosate by one of the two known routes (Moore et al., 1983; Talbot et al., 1984; Shinabarger and Braymer, 1986; Balthazor and Hallas, 1986; Kishore and Jacob, 1987; Wackett et al., 1987a; Pipke et al., 1987a; Pipke et al., 1987b; Hallas et al., 1988; Jacob et al., 1985 and 1988; Pipke and Amrhein, 1988; Quinn et al., 1988 and 1989; Lerbs et al., 1990; Schowanek and Verstraete, 1990; Weidhase et al., 1990; Liu et al., 1991). A route involving a “C-P lyase” that degrades glyphosate to sarcosine and inorganic orthophosphate (Pi) has been reported for a Pseudomonas sp. (Shinabarger and Braymer, 1986; Kishore and Jacob, 1987) and an Arthrobacter sp. (Pipke et al., 1987b). Pure cultures capable of degrading glyphosate to AMPA have been reported for a Flavobacterium sp. (Balthazor and Hallas, 1986), for a Pseudomonas sp. (Jacob et al., 1988) and for Arthrobacter atrocyaneus (Pipke and Amrhein, 1988). In addition, a large number of isolates that convert glyphosate to AMPA have been identified from industrial activated sludges that treat glyphosate wastes (Hallas et al., 1988). However, the number and nature of bacterial genes responsible for these degradations have not been heretofore determined nor have the gene(s) been isolated.
Hence, in one aspect, an object of the present invention is to provide novel genes which encode a glyphosate metabolizing enzyme which converts glyphosate to aminomethylphosphonate and glyoxylate.
Another object is to enhance the activity of the glyphosate metabolizing enzyme against glyphosate by replacement of specific amino acid residues.
Another object of the present invention is to provide genetically modified plants which express a gene which encodes a glyphosate metabolizing enzyme and which exhibit enhanced tolerance to glyphosate herbicide.
Another object is to demonstrate that a glyphosate metabolizing enzyme can be targeted to plastids using chloroplast transit peptides and the plastid targeted enzyme confers high level glyphosate tolerance.
A further object is to provide a method for selecting transformed plant tissue using the glyphosate metabolizing enzyme as the selectable marker in the presence of inhibitory concentrations of glyphosate.
These and other objects, aspects and features of the present invention will become evident to those skilled in the art from the following description and working examples.